1. Field of the Invention
The invention relates to a microbiological method for the biosynthesis of the natural blue-violet dyes violacein and deoxyviolacein from a pigment-forming bacterium by cultivation of the bacterium, removal of its cell mass by centrifuging, extraction of a raw dye extract from the cell mass and isolation of the dyes from the raw dye extract, and to the utilization of the dye produced by the microbiological method.
Dyes are of interest in more than one respect to microbiology and to chemistry. Since the ability to produce dyes is genetically fixed, the formation of dyes is also a first indication of the coincidental formation of antibiotic agents. Microbial pigments are of great structural diversity. They may be derivatives of the material classes of carotenoids, phenazine dyes, pyrrole dyes, azaquinones, etc.
2. The Prior Art
For about the past twenty years, there has been an ongoing search in the United States of America and in Japan, with thermophilic bacteria from deep sea vents and symbiontic bacteria from invertebrate and vertebrates, for biotechnologically useful bioactive natural substances. In countries of the European Community the usefulness of marine organisms has been recognized for the pharmaceutical industry, aqua culture, microbiological cleansing of oil contaminated areas of the ocean as well as for development processes of biological films and biological adhesion. However, in Germany it was not until three years ago that projects were conceived on a larger scale, which involve marine organisms in a more application-oriented direction of research. A large number of dyes are used in the textile, food, cosmetics and pharmaceutical industries. During recent years there has been a pronounced trend of consumers preferring products which contain natural substances. At present, yellow and red dyes are already being produced on an industrial scale from vegetable raw materials. However, in the mentioned industrial fields there exists a large demand for natural blue and violet dyes. The search for a dye from this spectrum has taken on a particular significance as a consequence of the legal prohibition against the use in foods of the red-blue dye monascine, isolated from Monascus purpureus. Classifying monascine as a food dye or colorant is not possible because of allergological problems. A microbiologically produced dye may thus help to close the gap and to open economically highly interesting paths.
Pigments from the blue-violet spectrum are formed, among others, by microorganisms. The azaquinone indigoidine is formed by several organisms (Pseudonomas indigofera, Corynebacterium insidiosum, Arthrobacter arthrocyaneus and Arthrobacter polychromogenes) and is secreted into the surrounding medium. This is of advantage in respect of a continuous process operation and of simplified product processing. It eliminates the step of cell pulping. Indigoidine has been officially approved as a food dye (E 132). Further approved food dyes from the blue dye spectrum are patent blue V (E 131), brilliant blue FCF and brilliant black (E 151). The absorption spectra of these compounds 11e in the range of 570 nm to 638 nm and are, therefore, particularly suitable for applications in food technology processes.
The blue-black pigment violacein, which is an indole derivative, was first described in literature in 1882. It was then that a violet dye was isolated from a bacterium which is now known as Chromobacterium violaceum. Heretofore, violacein could be isolated from the bacterial strains of Janthinobacterium lividum, Chromobacterium lividum and Alteromanus luteoviolacea (see H. Laatsch et al.: “Spectroscopic Properties of Violacein and Related Compounds: Crystal Structure of Tetramethylviolacein”; J. Chem. Soc. Perkin Trans 2., 1984, pp. 1331-1339). The structure of violacein was proved by synthesis in 1960. Chemically, violacein is characterized as 3-[1,2-dihydro-5-(5-hydroxy-1H-indole-3-yl)-2-oxo-3-H-pyrrole-3-ylidene]-1,3-dihydro-2H-indole-2-on, of the summation formula C20—H13—N3—O3 (molecular weight 343.33). The maximum absorption band, in a solution of methanol, is about 570 nm. In water, violacein is practically insoluble; but it is soluble in acetone, ethanol and dioxane. Violacein is the major component of a blue-violet pigment in Chromobacterium violaceum which is a microorganism taken from the soil and water of tropical regions, with deoxyviolacein occurring as a secondary component of identical structure, except that it has one less oxygen atom (summation formula C20—H13—N3—O2). So-called “native violacein” consists of a mixture of violacein with up to 10% deoxyviolacein. The violacein serves to protect the cell from radiation and to regulate the concentration of tryptophane below the toxic level. While violacein has antibiotic, antiviral and antitumoral properties, it displays no cyto-toxic or pathogenic effects. In more recent examinations, violacein has also been used for dying textiles. Good dying results have not only been obtained in connection with natural fibers such as silk, wool and cotton, but also with synthetic fibers such as polyamide (see Shirata et al.: “Isolation of Bacteria Producing Bluish-Purple Pigment and Use for Dying”, Jpn. Agr. Res. Q 2000, 34(2), pp. 131-140, using pigment produced from Janthinobacterium lividum).
The biosynthesis from known microorganisms has been described in various publications. A method of producing native violacein, proceeding from the pigment-forming bacterium Chromobacterium violaceum, for use in the treatment of viral diseases, is described in German patent specification DE 3,935,066. The biosynthesis is based on the method steps of “cultivating the bacterium”, “removing the cell mass by centrifuging”, “extracting a raw dye extract from the cell mass”, and “isolating the dye from the raw dye extract”. The bacteria used as the starter material are cultivated on a solid or liquid nutrient. Preferably, the bacteria are cultivated in liquid nutrients since they can then be easily separated from their nutrient by centrifuging. The cultivation of the bacteria may be carried out with different process parameters in fermentation tanks. In the known method, the grown bacterial bed, after incubation, is separated and freeze-dried. Extraction of the raw violacein is carried out in methanol in a Soxhlet extractor. The methanol is removed by vacuum distillation. For isolating the violacein, the raw violacein is twice extracted by n-heptane, and is then filtered out. The residue is dissolved in a mixture of chloroform, acetone, pyridine 50:40:10. The mixture of the dye is then separated and purified by silica gel thin layer chromatography.
A simple method of obtaining highly purified violacein is known from the paper “Production, Extraction and Purification of Violacein: An Antibiotic Pigment Produced by Chromobacterium violaceum (Rettori, Duran, World Journal of Microbiology & Biotechnology 14 (1998), pp. 685-688. To cultivate the bacterium, non-sterile cotton rags are inoculated with a suspension culture of the pigment-forming Chromobacterium violaceum CCT 3496 and are stored in a strongly ventilated incubator where strong growth of the bacterium occurs. Thereafter, the cell mass is washed out of the cotton rags with a solvent, and is filtered to yield an extract of raw dye. A highly purified violacein is isolated from this as a dye by different methods (Soxhlet extractor, high-performance liquid chromatography). However, the yield is relatively low and lies in the range of milligrams per liter of nutrient.
The production of violacein from L-tryptophan as a biosynthetic starter material is known from the paper “Biosynthesis of Violacein: A Novel Rearrangement in Tryptophan Metabolism with 1,2-shift of the Indole Ring” (T. Hoshino et al., Agric. Biol. Chem. 51 (3), 1987, pp. 965-968, wherein the carbon skeleton of the pyrroleinone ring is formed by condensation of the side groups of two tryptophan molecules by a 1,2-shift of the indole ring. By comparison with the production of violacein from a pure suspension culture of Chromobacterium violaceum JCM 1249, it is possible to obtain an about 1.5-fold yield by adding L-tryptophan to the suspension culture. However, at the usual yield in the range of milligrams per liter of nutrient, this is still to be considered as a very low yield.
In summary, the known methods of biosynthesis of violacein and deoxyviolacein are not suitable for providing these dyes in greater quantities. For use in the pharmaceutical field this is not absolutely necessary, since in this context even the smallest quantities may be effectively applied. In other applications, however, the industrial demand for blue-violet dyes may be very large. If they are synthesized by the known methods, the use of large quantities of suspension cultures is necessary, the processing of which will be correspondingly demanding as regards equipment and time.